This invention relates to a method for detecting a smell-emitting substance produced owing to ageing, by using Y-shaped labyrinth or GC.
Experimental Animals and Method for Collecting Urine
Mice (C57BL/6J-H-2-k) were used as smell sensors (n=5) for Y-shaped labyrinth and as experimental animals for estimating the change of the body smell with ageing. The mice were divided into three groups (n=15-30), i.e., a group of young mice (1-3 months), a group of adult mice (3-10 months) and a group of aged mice ( greater than 17 months). Urine was used as a smell source. Urine was collected from each group by lightly pressing the bladders of the mice so as to minimize a stress imposed to the mice. Each urine sample is a mixture of urines collected from 2 to 6 mice in the same group. Each sample was stored at xe2x88x9220xc2x0 C.
Preparation of Samples for Y-shaped Labyrinth Method and Gas Chromatography
Ether extract fractions of the urine samples were/prepared according to the method described in reference 1. Each urine sample was subjected to centrifugal filtration at 5xc2x0 C. and 5,000xc3x97g (6,500 rpm) by the use of Centricon-10 (Amicon, 10,000 MW cut off) to remove urinary proteins such as major urinary proteins. Then, 1.0 ml of the filtered urine sample was saturated with 200 mg of KH2PO4 to obtain a weekly acidic fluid (pH 4.4-4.6). This fluid was extracted by shaking for 2 hours with diethyl ether (HPLC grade) 15 times volume as much as the fluid. The extract solution was dispensed in 7.5-ml aliquots into test tubes, after which the extract solution in each tube was concentrated to 0.5 ml with Speed Vac (60 min) and freeze-dried for about 70 minutes.
Y-shaped Labyrinth Method (Involving Live Animals in a Maze)
The change of the body smell with ageing was estimated by Y-shaped labyrinth method according to the method described in reference 2. FIG. 1 shows a general view of Y-shaped labyrinth. In Y-shaped labyrinth method, a urine sample is placed in a box located at an end of the two branches of the Y-shaped labyrinth, and another urine sample is placed in the other box located at the other end of the two branches of the Y-shaped labyrinth. The two urine samples are from two groups of mice that belong to the same strain but are at different ages. The mice are trained to select the smell of one of the urine samples. The mice have been fed with a sufficient diet but have been given a limited volume of water. They are given a drop of water as a reward only when they select the correct smell. If the mice can distinguish the smells, this discrimination proves that the smell given out by the animal varies depending on the age of the animal. Although the percentage of correct answers by the trained mice is about 50% for first several days, it increases gradually and exceeds 80%. In the present example, the mice were trained at first to distinguish the 5-fold dilutions of urine samples of the group of adult mice from those of the group of aged mice. Then, whether the mice could distinguish the ether extracts of urine samples was confirmed. Lastly, whether the mice could distinguish the ether extracts of different urine samples without being rewarded with water was observed.
Conditions of Gas Chromatography
Gas chromatography was carried out by using Varian 3300. In the experiments, a Stabilwax column (30 mxc3x970.32 mm i.d.xc2x710 xcexcm df) was used as a column for separation after having been equipped with a 5 mxc3x970.53 mm i.d. guard column (Restek, Bellefonte, Pa.). Solutions prepared by re-dissolving the ether extract of the weekly acidic urine in methyl acetate were used as samples as described above. Fractions composed mainly of any of volatile organic acids, amines, amides and the like in the urine samples from the mice were identified. The column temperature was maintained at 80xc2x0 C. for 2 minutes and then raised to 240xc2x0 C. at a rate of 5xc2x0 C. per minute. Helium was used as a carrier gas and the linear velocity was adjusted to 37 cm/sec (at a column temperature of 40xc2x0 C.). Quantitative analysis of the components on the chromatogram was conducted according to peak percentage method.